Particulate compositions for chemical synthesis

ABSTRACT

Disclosed are compositions that include triboelectrically chargeable nucleotide particles of less than 50 μm diameter and carrier particles. In one example, a substrate is selectively patterned with the compositions, e.g., by transfer from a selectively charged surface. The compositions can be used to synthesize nucleic acid arrays.

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims benefit or priority to U.S. ProvisionalPatent Application Serial No. 60/279,004, filed Mar. 26, 2001, andapplication Serial No. 60/322,362, filed Sep. 14, 2001, the contents ofboth of which are incorporated herein by reference in their entirety.

BACKGROUND OF THE INVENTION

[0002] The invention relates to the synthesis of arrays of chemicalcompounds. A variety of chemical compounds can be synthesized on a solidsupport. Combinatorial libraries of chemical compounds can besynthesized on the solid phase (see, e.g., DeWitt et al. (1993) Proc NatAcad USA 90:6909; and Bunin et al. (1994) 91:4708). For example,Merrifield developed a method for synthesizing peptides on a support(Merrifield (1965) Science 150:178-185). This method was extended tooligonucleotides by Letsinger and others (see, e.g., Beaucage and Iyer(1992) Tetrahedron 12:2223 for a review). Arrays of chemical compoundscan be produced by synthesizing the different compounds on a commonsolid support, e.g., a planar surface of the support. Such arraysprovide a convenient format for analyzing multiple interactions inparallel, and, accordingly, is adaptable for high throughput screening,e.g., for drug discovery, gene discovery, genotyping, and diagnosis.

[0003] Arrays can be produced by the in situ synthesis of compounds onthe solid support to form the covalent immobilization of compounds arein-situ synthesis of biopolymers from properly protected monomers.

[0004] Photolithographic methods (see, e.g., Fodor et al. (1991) Science251:767-773; Fodor et al. (1993) Nature 364:555-556; U.S. Pat. No.5,143,854) have been used to construct oligonucleotide arrays withspatial addressability. This approach, at least in some implementations,uses customized photomasks to control the addition of a particularnucleotide monomer at each successive addition reaction. In anotherapproach, micromirrors (see, e.g., Gao et al. (2000) J. Comb. Chem. 2:349-354; M. R. Sussman et al. (1999) Nature Biotechnology 17:974-978;and WO 99/41007) are employed to provide an addressable array of lightwithout the use of a photomask. The light can locally produce acid whichsubsequently removes the acid labile protecting groups,4,4′-dimethoxy-trityl (DMT), on nucleotide monomers.

[0005] A third approach uses a pulse jet, such as found in an inkjetprinter, to distribute sub-nanoliter volume of nucleotide monomersolution and the activation agent such as tetrazole (see, e.g., WO95/25116, U.S. Pat. Nos. 6,028,189; 5,874,554; 5,474,796; 6,177,558 andWO 98/41531). The piezoelectric pumps deliver, e.g., 5′-protected,3′-phosphoramidite activated nucleotides dissolved in organic solvent toindividual spots on an array. The solution of activated nucleotide ismixed with a solution of a coupling agent to covalently attach theactivated nucleotide to a functional group on the glass surface. The5′-protecting groups are subsequently removed with a deprotectingreagent. The entire process is repeated until the desiredoligonucleotides are formed.

[0006] In one implementation, five nozzle heads are used for DNAsynthesis using the pulse jet method. Four nozzle heads are provided forthe four nucleotide monomers A, T, G, C, and a fifth nozzle head is usedto deliver a solution of an activating agent for coupling reactionsafter each nucleotide monomer is printed on the substrate. The accuracyof delivering the activating agent to a location where one of thenucleotide monomers is previously spotted can impact the couplingreaction (see, e.g., U.S. Pat. Nos. 5,985,551 and 5,474,796). The entireapparatus is kept in a moisture-free environment at every step in theprocess.

SUMMARY

[0007] The present invention provides methods of dispensing the nucleicacid subunits as a dry composition (e.g., a particulate composition) forthe in-situ synthesis of nucleic acid polymers. Because the drynucleotide are chemically stable, deposition of the four nucleotidemonomers in dry form can be accomplished simultaneously on the samesubstrate in an open environment, thus, facilitating the synthesis ofthe biopolymer on a substrate surface. A subunit for polymer synthesisis a compound that can be used to construct a polymer such that at leasta portion of the subunit becomes incorporated in the polymer. A typicalsubunit is a monomer subunit, e.g., a nucleotide for nucleic acidsynthesis. Larger subunits that can be used are dinucleotides andoligonucleotiders (e.g., heptamers, and so forth).

[0008] In one aspect, the invention features a composition, e.g., atoner composition, that includes chargeable particles of diameter lessthan about 100, 50, 20, 18, 15, 12, 10, 9, 8, 7, or 6 μm. The particlesinclude a compound that has a reactive group and a protecting group. Theparticles can be prepared with additives to improve the physicalproperties such as triboelectrification. The composition can furtherinclude chargeable beads of at least 30, 40, 50, 60, or up to 200 μm indiameter. The chargeable beads can be larger in diameter than thechargeable particles.

[0009] In some embodiment, the compound is a nucleotide or an aminoacid. A nucleotide compound can have the protecting group attached atthe C-3′ or the C-5′. The nucleotide compound can have a reactive groupthat is phosphoramidite. An amino acid compound can have the protectinggroup attached to its amino or carboxyl group. The protecting group canbe photosensitive, acid-sensitive, or base-sensitive. In some instances,the compound may be a preformed polymer, e.g., a protected dinucleotideor other oligonucleotide that includes a protecting group.

[0010] In one aspect, the invention features a composition thatincludes: particles of less than 100, 50, 30, 20, 10, 7, or 5 μmdiameter, the particles are composed at least partially of a nucleotidethat includes a 5′ and 3′ functional groups (e.g., a protecting groupand a phosphoramidite). The particles can be at least 0.02. 0.05, 0.1,or 0.2 μm in diameter. Typically, one of the functional groups is aprotecting group and the other is a reactive group. All nucleotideparticles of the composition may be of less than 50 μm diameter. Forexample, the composition can be devoid of nuclceotide particles ofgreater than 10, 20, 30 or 50 μm diameter. The composition can be a drycomposition or a liquid composition.

[0011] In one embodiment, the nucleotide particles are between 0.1% and100%, e.g., 0.1% and 50%, or 1% and 95% of the composition by weight.Viewed, as a percentage weight of the total nucleotide weight in thecomposition, the nucleotide particles of the given diameter can be atleast 20, 40, 50, 60, or 80% of the composition.

[0012] The nucleotide can be, e.g., adenine, guanine, cytosine, orthymidine.

[0013] In another aspect, the invention features a composition thatincludes: chargeable particles (e.g., triboelectrically chargeableparticles) of less than 50 μm diameter, and carrier particles. Thechargeable particles are at least partially composed (e.g., at least 5,10, 20, 50, 80%) of nucleotide. The composition can be a drycomposition.

[0014] The nucleotide particles can be positively and/or negativelychargeable to 5 to 50 μC/g or 10 to 30 μC/g, or about 1 to 10 μC/g. Thecarrier particles can include a metal oxide, e.g., one or more offerrite, zinc oxide, and nickel oxide. Agitation of the composition cancharge the nucleotide particles. The composition itself can be chargedor uncharged at a given instance.

[0015] The composition can include a surface charge control agent suchas polystyrene, polyformaldehyde, polymethylmethacrylate, polyamide,fumed silica, cabosil, amorphous silica, a fluorinated polymer (e.g.,Kynar), polyethylene glycol succinate, polyethylene glycol adipate,polydiallyl phthalate, polyurethane elastomer, styrene-acrylonitrilecopolymer, styrene-butadiene copolymer, polyisobutylene, borosilicateglass, polyethylene glycol terephthalate, epoxide resin,polychlorobutadiene, butadiene-acrylonitrile copolymer,polyacrylonitrile, polyethylene, chlorinated polyether, zinc stericpolytrifluorochloroethylene and polytetrafluoroethylene.

[0016] In another aspect, the invention features a composition thatincludes: particles of less than 50, 30, or 10 μm diameter, theparticles being at least partially composed of a nucleotide, means forcontrolling surface charge on the particles; and means fortriboelectrically charging the particles.

[0017] In still another aspect, the invention includes a compositionthat includes: a dielectric liquid component and particles. Theparticles are insoluble in the liquid and are composed of a protectedchemical compound, e.g., a nucleotide subunit. The particles of thecomposition are less than 50, 30, 20, 10, or 7 μm in diameter. Theparticles can be, for example, between 1 and 40% of the composition,weight to volume. In one embodiment, the dielectric liquid hasresistivity of between 10⁸ Ωcm and 10¹² Ωcm. The dielectric liquid canbe at least partially composed (e.g., at least 80%, 95%, 100%) of achemically inert organic solvent, e.g., a high molecular weightaliphatic hydrocarbon solvent such as an isoparaffin. The compositioncan further include a means for stabilizing charge.

[0018] In another aspect, the invention features a method that includes:providing a solid composition composed of a nucleotide monomer subunitthat is activated at one terminus and protected at another terminus;processing the solid composition to produce nucleotide particles thatare less than 50 μm in average diameter; and combining the nucleotideparticles with a carrier agent (e.g., carrier beads such as ferritecarrier beads) to produce a developer composition. In one embodiment,the nucleotide particles are at least 5, 10, 30, 50% of the solidcomposition by weight.

[0019] The processing can include jet milling. The processing canfurther include isolating particles that are less than 10 μm indiameter. Hence, in some embodiments, the processing produces nucleotideparticles that are less than 20, 10, 7, or 5 μm in diameter.

[0020] The method can further include combining the nucleotide particleswith a surface charge control agent, e.g., an agent described herein,including one or more of: cabosil, fumed silica, a fluorinated polymer,and polystyrene.

[0021] In another aspect, the invention features a method that includes:providing a particulate composition (e.g., a toner composition) thatincludes particles of a chemical compound; triboelectrically chargingthe particles (e.g., by agitation); contacting the charged particles toa selectively charged surface; and transferring the nucleotide particlesfrom the surface to a substrate. The particulate composition includes:(a) chargeable particles of less than 50 microns diameter, the particlesinclude a chemical compound, e.g., a compound that has an activatedgroup and/or a protecting group, and (b) chargeable carrier beads. Thechemical compound can be, e.g., a nucleotide, an oligonucleotide, or anamino acid. In one example, the particles are composed of a nucleotidethat has a phosphoramidite group and a protecting group

[0022] The selectively charged surface can include discrete areas thatare electrostatically repel the charged nucleotide particles and otherareas to which the charged nucleotide particles attach. The selectivelycharged surface can be cylindrical, e.g., a drum, or can benon-cylindrical (e.g., a belt). The selectively chargeable surface canbe a surface whose charge is altered by illumination, e.g., aphotoreceptor.

[0023] In one embodiment, the agitating charges the nucleotide particlesto between 5 μC/g to 50 μC/g. The toner composition can further includea surface charge control agent.

[0024] In another aspect, the invention features a method of coupling acompound to a substrate that has an immobilized reactive group. Themethod includes forming a dry thin film of a compound having a reactivegroup and a protecting group on the substrate; and contacting an organicsolvent having an activator compound dissolved therein to the substrateto thereby couple the compound to the substrate by coupling theimmobilized reactive group to the compound reactive group. An activatorcompound is a compound that is able to activate a phosphorous activatinggroup such as a phosphoramidite to be come a highly reactivephosphitilating agent so that a nucleotide that includes the phosphorousactivating group covalently couples to a receiving group. The activatorcompound can be a weak acid. Preferably, the activator compound resultsin near quantitative yield. For example, the activator compound can betetrazole, 5-(p-nitrophenyl)-1H-tetrazole, 5-ethylthio-1H-tetrazole,4,5-dichloroimidazole, benzimidazolium triflate, and 4,5dicyano-imidazole.

[0025] In still another aspect, the invention features a method ofdepositing a chemical compound. The method includes: (1) combining atoner composition that includes chargeable particles of a compound thathas a reactive group and a protecting group (e.g., as described herein)with charged carrier beads to generate developer particles; (2)depositing the compound from the developer particles in a loading zoneonto a surface region of a photoreceptor, the surface region beingselectively activated by illumination; (3) positioning the photoreceptorto displace the surface region from the loading zone into proximity toor contact with a substrate (e.g., via a charge device); and (4)transferring the compound to the substrate.

[0026] The compound can be a nucleotide, an oligonucleotide, an aminoacid, or another small organic molecule. The method can includerepeating steps (1) to (4) for a second compound, e.g., the compound canbe different from the first compound.

[0027] The method can further include (5) contacting a solventcontaining an activator compound to the target substrate, such that theliquid activator agent dissolves the transferred dry compound, catalyzesthe coupling of the reactive group of the protected chemical compound toan immobilized group on the target substrate. For example, the activatorcompound can be tetrazole, 5-(p-nitrophenyl)-1H-tetrazole,5-ethylthio-1H-tetrazole, 4,5-dichloroimidazole, benzimidazoliumtriflate, and 4,5 dicyano-imidazole. The solvent can be an aproticorganic solvent, e.g., acetonitrile, dinitriles and other solventmentioned herein. The organic solvent can be a high-boiling pointsolvent.

[0028] In a method of synthesizing nucleic acids, the method can includerepeating steps (1) to (4) for each of four nucleotides. The method canfurther include optionally (6) contacting the target substrate with acapping reagent, e.g., acetic anhydride in pyridine; optionally (7)oxidation; and (8) exposing the target substrate to a condition thatreleases the protecting group from the immobilized compound. The methodcan then include the addition of the next nucleotide to each of thenucleic acids on the target substrate by repeating the method until thearray of nucleic acids is complete.

[0029] In another aspect, the invention features a method ofconstructing a nucleic acid array. The method includes: determining aplurality of layer maps from a set of nucleic acid sequences, each layermap corresponding to a particular register and nucleic acid subunit andindicating discrete addresses at which the corresponding nucleic acidsubunit is to be coupled; for each register, directing a plurality ofapplicator units that each are supplied with a different nucleic acidsubunit to each transfer the respective nucleic acid subunit to asubstrate at the discrete addresses indicated by the layer map for therespective nucleic acid subunit; and coupling the transferred nucleicacid subunits to a reactive terminus to form an array of nucleic acids.

[0030] In one embodiment, the reactive terminus is on the surface of thesubstrate, e.g., the substrate is the target substrate. In anotherembodiment, the reactive terminus is on the surface of a secondsubstrate, and the nucleic acid subunits are transferred from the firstsubstrate (e.g., an interim substrate) to the second substrate (e.g.,the target substrate).

[0031] The nucleic acid subunits can be supplied as particles of lessthan 50 μm diameter, e.g., a composition described herein. The subunitcan be a monomeric subunit.

[0032] In one embodiment, each applicator unit includes a photoreceptor,an illumination unit that selectively alters the charge at discretepositions on the photoreceptor, and an agitator that agitates a tonercomposition comprised of the nucleic acid subunit particles and carrierbeads.

[0033] In another aspect, the invention features a method thatincludes: 1) repeating for each of four nucleotide bases, adenine,guanine, cytosine, and thymidine,

[0034] a) triboelectrically charging particles of the respectivenucleotide base, the base including a protecting group;

[0035] b) selectively irradiating a photoreceptor to generate apatterned region with defined electrostatic charge;

[0036] c) contacting the charged nucleotide particles to thephotoreceptor to attach the particles to the photoreceptor in thepatterned region; and

[0037] d) transferring the nucleotide particles from the photoreceptorto a surface;

[0038] 2) coupling the transferred particles to terminal groups on asolid support; and 3) repeating 1) and 2) to produce a plurality ofdifferent nucleic acid sequences on the solid support. The method can beused to provide a nucleic acid array.

[0039] In one embodiment, the surface is the surface of an interimsubstrate and the method further includes transferring the particlesfrom the surface of the interim substrate to the solid support. Forexample, a different interim substrate can be used for each repetitionof 1) and 2).

[0040] In one embodiment, the coupling comprises contacting theparticles to an activator compound, e.g., an activator compounddescribed herein.

[0041] The cycles of a) to d) can include conveying the substrate (e.g.,a flexible substrate) between each of four applicator units, eachapplicator unit comprising a photoreceptor and a source of therespective nucleotide particles. The substrate can include paper, Mylar,cellulose, polyvinylchloride, and/or polycarbonate.

[0042] In another aspect, the invention features an apparatus fordepositing chemical compounds on a substrate. The apparatus includes asubstrate path, adapted to move a substrate; applicator units; an imageroll that includes a photoreceptor surface, a transfer unit, and achemical fuser. The transfer unit can include a electrical fieldgenerator, e.g., a scanning electrical field generator. The transferunit can include support for positioning an interim substrate inapposition to a target substrate.

[0043] The substrate path can be adapted to move the substrate e.g.,from the image roll to the transfer unit. Each applicator unit caninclude a charge pattern generator and a dispenser that deposits chargedparticles, for example, particles of a chemical compound having aprotecting group and a reactive group. The charge pattern generator caninclude a charger which deposits electrostatic charge on thephotoreceptor surface and a light source which selectively illuminatesthe photoreceptor surface to generate a pattern of electrostatic charge(e.g., a latent image). The light source can be interfaced to acontroller, e.g., a computer system programmed with a method describedherein. The source can be, for example, a laser or a light-emittingdiode array.

[0044] The image roll is configured to rotate regions, of thephotoreceptor surface between the applicator units and the substrate.For example, the image roll can be a drum or a belt that moves regionsof the photoreceptor surface between applicator units and the substrate.The photoreceptor surface is selectively chargeable by light dischargedfrom the charge pattern generator for adherence to the charged particlesdeposited by the dispenser.

[0045] The fuser can be configured to dispense an organic solution or anaerosol thereof onto the target substrate. The organic solution containsan activator compound.

[0046] The apparatus can include, for example, 2, 3, 4, 5, 6, 9, 10, 18,19, 20, 21, 22 or more applicator units. An apparatus adapted tosynthesize nucleic acids on a substrate can, for example, have at leastfour applicator units. An apparatus adapted to synthesize peptides on asubstrate can, for example, have about twenty applicator units (e.g., atleast ten, fifteen, or nineteen). Further, the apparatus can be adaptedto have two or more image rolls, and the applicator units can bedistributed among the image rolls. For example, an apparatus adapted tosynthesize nucleic acid on a substrate can have two image rolls, eachhaving two applicator units.

[0047] In another aspect, the invention features an apparatus fordepositing chemical compounds on a substrate. The apparatus includes asubstrate path, adapted to move a substrate; photoreceptors, applicatorunits; registration unit and a chemical fuser. Each photoreceptor isadapted to move a region of its surface from a loading zone of anapplicator unit to a position along the substrate path such that thesurface can contact a substrate on the path. For example, thephotoreceptor can be a cylinder or drum that rotates regions of itssurface between the loading zone and the substrate path. In anotherexample, the photoreceptor is a belt.

[0048] Each applicator unit can include a charge pattern generator and adispenser that deposits charged particles, for example, particles of achemical compound having a protecting group and a reactive group. Thecharge pattern generator can include a charger which depositselectrostatic charge on the photoreceptor surface and a light emitterwhich selectively illuminates the photoreceptor surface to generate apattern of electrostatic charge. The light emitter can be interfaced toa controller, e.g., a computer system programmed with a method describedherein.

[0049] The dispenser is configured to combine, e.g., with brush, blades,or motors, a toner composition with the carrier beads, and to depositdeveloper particles that include the carrier beads and a tonercomposition onto a photoreceptor.

[0050] The fuser is adapted to dispense an organic solvent (e.g.,containing an activator compound) onto the substrate, as described aboveand herein.

[0051] For example, when adapted for nucleic acid synthesis, theapparatus can include four photoreceptor, each having an applicatorunit. The substrate path can position the substrate at each of the fourphotoreceptors, to receive sequentially all four possible subunitnucleotides. The path can then position the target substrate at thechemical fuser and other optionally post-processing stations, and returnthe substrate back to the photoreceptors to repeat the process.

[0052] In the case of oligonucleotide arrays, only four applicatorunits, one for each base, are required to produce arrays with flexiblesequence composition and spatial arrangement. Unlike the deposition ofpreformed oligonucleotides, the same four applicators are used in acyclic manner (e.g., without cleansing and exchange of reagents) toproduce any sequence formed from the four canonical bases. As seen, eachapplicator is dedicated to the deposition of a particular nucleotide(e.g., a nucleotide selected from the deoxyribonucleotides: adenine,guanine, cytosine and thymidine, or in some cases ribonucleotides, inwhich case uracil may be used).

[0053] Although the simple four applicator design is completelyversatile for most purposes, additional applicators can be used to adddinucleotides and larger oligomers that are protected and that can becoupled to growing chains, and/or unnatural nucleotides that areprotected. Kumar and Pumian (1984) J. Org. Chem. 12:3387, e.g., describecoupling with phosphoramidite activated dinucleotides. For example, ifan array of primers is being constructed such that the primers allinclude a universal sequence, the universal sequence can bepresynthesized in bulk on a bead using a conventional automatedoligonucleotide synthesizer. The oligonucleotide is protected and/orphosphoramidite activated and added to a “fifth” applicator unit. Withrespect to applications that require an unnatural nucleotide, examplesof such unnatural nucleotides include inosine, abasic nucleotides, andnucleotides that include nitro or cyano substituted indoles, (e.g.,3-nitropyrrole nucleotide), e.g., as described in U.S. Pat. No.5,780,223.

[0054] In another aspect, the invention features a method that includes:depositing a nucleotide compound as a particulate composition, thenucleotide compound having a first functional group on a solid supportthat includes a second functional group attached thereto, and contactingthe solid support with an aerosol of a liquid composition that includes,dissolved therein, an activator compound that triggers the coupling ofthe first functional group to the second functional group. Exemplaryactivator compounds include: tetrazole, and derivatives thereof, e.g.,5-(p-nitrophenyl)-1H-tetrazole, 5-ethylthio-1H-tetrazole,4,5-dichloroimidazole, benzimidazolium triflate, or 4,5dicyanoimidazole. The concentration of the activator compound in theorganic solvent can be between 0.1 M and 1.0 M.

[0055] In one embodiment, the particulate composition is dry at theinstance of the depositing; in another, the particulate composition issuspended in a dielectric liquid (e.g., a chemically inert liquid suchas an isoparaffin) at the instance of the depositing.

[0056] The liquid composition that is aerosolized can include a highboiling point organic solvent, e.g., propylene carbonate, a dinitrile,or diethyl carbonate. In one embodiment, the liquid composition is amixture that includes high boiling point organic solvent and a lowboiling point organic solvent, e.g., an aprotic solvent, such asmethylene chloride or acetonitrile. In one embodiment, the mixture isbetween 20:80 and 80:20, or 40:60 to 60:40. One exemplary mixtureincludes propylene carbonate and acetonitrile.

[0057] In one embodiment, the nucleotide compound further comprises aprotecting group, attached to C-3′ or C-5′ of the nucleotide residue.The compound can include a phosphoramidite at the other terminus. Inanother example, the compound includes a hydroxyl group at the otherterminus. The protecting group can be photo, acid, or base sensitive.

[0058] In one embodiment, the method further includes reacting uncoupledsecond functional groups with a capping reagent, e.g., an acylatingreagent, e.g., acetic anhydride.

[0059] In another aspect, the invention features a method that includes:depositing, on a solid support, different nucleic acid subunits atdifferent addresses, and contacting a section of the solid support withan aerosol of an organic solvent that includes an activator reagentdissolved therein. At least a plurality of addresses within the sectioncan be concurrently contacted by the aerosol. The activator reagenttriggers the coupling of the nucleic acid subunit to a group attached tothe support. The long axis of the contacted section can be at least 0.02meters, e.g., a section of at least 0.04 by 0.04 meters, 0.04 by 0.1meters, or 0.1 by 0.15 meters. The nucleic acid subunit can include aprotecting group.

[0060] In one embodiment, the nucleic acid subunits are dissolved in alow boiling point solvent. The method can include evaporating the lowboiling point solvent prior to contacting the solid support with anaerosol of organic solvent, e.g., to form a dry thin film is formed bythe evaporating. In another embodiment, the nucleic acid subunits aredeposited as a particulate composition.

[0061] In another aspect, the invention features a method that includes:depositing a nucleotide compound dissolved in a low boiling pointsolvent, the nucleotide compound having a first functional group, on asolid support that includes a second functional group attached thereto,and contacting the solid support with an aerosol of a liquid compositionthat includes, dissolved therein, an activator compound that triggersthe coupling of the first functional group to the second functionalgroup, wherein the liquid composition is a mixture of a low boilingpoint solvent and a high boiling point solvent.

[0062] In still another aspect, the invention features a method thatincludes: forming a dry deposition (e.g., a thin film or a particulatedeposition) of a compound (e.g., a nucleotide residue) having a firstreactive group (e.g., a hydroxyl or phosphoramidite) on a substrate,wherein the substrate has an immobilized second reactive group, andcontacting an organic solvent to the substrate, the solvent having anactivator compound dissolved therein, thereby immobilizing the compoundto the substrate by coupling the first and second reactive groups. Thenucleotide residue can include a C-3′ or C-5′ protecting group.

[0063] In one embodiment, the method further includes reacting uncoupledsecond reactive groups with a capping reagent.

[0064] The forming can include depositing the compound dissolved in alow boiling point solvent onto the substrate, and evaporating the lowboiling point solvent.

[0065] In one embodiment, the first reactive group is a phosphoramidite.The second reactive group can be a hydroxyl.

[0066] In one embodiment, the invention features a method ofimmobilizing a compound to a solid support. A compound having a reactivegroup is deposited onto a solid support having an immobilized functionalgroup. The substrate is then contacted with an aerosol of an activatorsolution wherein the activator solution has an activator reagentdissolved in a solvent. Upon contact with the activator solution, thecompound becomes attached to the substrate by coupling of the functionalgroup with the activator group.

[0067] The method can further include immobilizing the compound wherethe compound is a solid. For example, the compound can be dry, i.e., notdissolved in solvent. One type of dry compound is provided in the formof a particulate composition. The compound can also be in the form of athin film on the solid support.

[0068] The compound can be a nucleotide, an amine acid, or a smallorganic molecule.

[0069] The compound can further include a protecting group. For example,the compound can be a nucleotide with a protecting group attached to theC-3′ or the C-5′. In another embodiment, the protecting group can beattached to an amino or carboxyl group of an amino acid. Further, theprotecting group can be photosensitive, acid-sensitive, orbase-sensitive.

[0070] In certain embodiments, the reactive group can be aphosphoramidite.

[0071] In certain embodiments, the functional group immobilized on thesolid support can be a hydroxyl group.

[0072] The activator compound can be tetrazole,5-(p-nitrophenyl)-1H-tetrazole, 5-ethylthio-1H-tetrazole,4,5-dichloroimidazole, benzimidazolium triflate, or 4,5dicyanoimidazole. The concentration of the activator compound can bebetween 0.1M and 1.0M, e.g., 0.2M to 0.8M or 0.2M to 0.6M. The activatorcompound can be dissolved in a high boiling point solvent, or a liquidcomposition that includes a high boiling point solvent and a low boilingpoint solvent.

[0073] The invention can also feature reacting unreacted immobilizedfunctional groups with a capping reagent. For example, the unreactedfunctional groups can be treated with an acylating agent such as aceticanhydride.

[0074] In another embodiment, the invention features a method of formingan array of compounds. The method includes immobilizing a dry compoundhaving a reactive group onto a solid support, wherein the solid supporthas attached functional groups. An organic solution of activatorcompound is then dispensed onto the solid support. This immobilizes thecompound to the solid support by coupling the reactive group with thefunctional group. The uncoupled functional groups are then reacted witha capping reagent.

[0075] The invention further features a method of coupling compounds bygenerating an aerosol of activating compound and contacting it with afunctional group and a dry composition that includes a protecting groupand an activating group. This causes coupling of the dry composition tothe functional group.

[0076] The method can further include generating the aerosol usingpositive pressure. The aerosol can also be generated from a spray headconnected to pressurized nitrogen. Additionally, the aerosol can begenerated into atmospheric conditions or non-atmospheric conditions.

[0077] In another embodiment, the invention features a method providinga solid support having a substrate with functional groups attached. Drythin films of compounds having a reactive group and a protecting groupare formed onto the solid support. The solid support is then contactedwith an aerosol of organic solvent, the solvent having an activatorcompound dissolved therein. This immobilizes the compound to thesubstrate by coupling the reactive group with the functional group. Theunreacted substrate is then reacted with a capping reagent, and theprotecting groups are subsequently removed. In another embodiment, themethod can be performed, repeating as many times as desired, the stepsfrom forming dry films of compounds onto the solid support to removingthe protecting groups.

[0078] The invention also features an apparatus. The apparatus includesapplicator units, each unit including a dispenser that depositscompounds onto a solid surface, a reservoir for storing an organicsolution comprising an activating reagent, and a spray head in fluidconnection with the reservoir and adapted to create an aerosol of theorganic solution. The apparatus can include an actuator that actuatesthe solid surface from the applicator units to a position to receive theaerosol from the spray head. The applicator units can each apply adifferent monomer, e.g., a nucleotide dissolved in a low boiling pointsolvent.

[0079] The apparatus can further include an applicator for cappingunreacted portions of solid support, wherein the applicator deposits thecapping reagent onto the solid support.

[0080] The apparatus can further include a housing adapted to provide adry atmospheric environment.

[0081] In another embodiment, the invention features a method of formingan array. The method includes: providing a planar solid support that hasa reactive surface; patterning each of a plurality of selective regionsof the solid support with a compound; covalently bonding the compound tothe support; and capping unpatterned regions of the support with anprotecting group, whereby the capping defines the shape of the regionsof compound on the solid support. The method is used during theformation of the first layer of the array, e.g., to pattern it forsubsequent layers. The planar solid support can have a uniform reactivesurface when provided. The compound can differ between selective regionsof the plurality.

[0082] In the context of this invention, the following terms aredefined: “Dry” means solid phase at a given time, i.e. not dissolved inan aqueous or organic solution. For example, a dry film of a nucleotidecan be made by applying a nucleotide dissolved in acetonitrile andevaporating the acetonitrile. “Aerosol” means a fine mist of a liquid.“Low boiling point solvent” means a solvent having a boiling point of85° C. or less at atmospheric pressure. A “high boiling point solvent”refers to a solvent that has a boiling point of greater than 85° C. atatmospheric pressure.

[0083] In another aspect, the invention features a method that includesforming a patterned deposition of a compound on a first substrate;positioning the first substrate in apposition to a second substrate; andtransferring at least a portion of the dry deposition from the firstsubstrate to the second substrate to produce a patterned dry depositionof the nucleotide on the second substrate. The patterned deposition canbe a dry deposition, e.g., a deposition of particles (e.g., particlesdescribed herein, such as nucleotide particles) or a thin film (e.g., athin film described herein). In one embodiment, the dry depositionincludes particles that include a nucleotide, e.g., a chargednucleotide. The nucleotide can include a 5′ or 3′ protecting groupand/or an activated group, e.g., a phosphoramidite.

[0084] The first substrate include sections that include depositions ofa first nucleotide and other sections that include depositions of asecond nucleotide, e.g., at least a section for each of at least fournucleotides.

[0085] The transferring can include applying an electrical field, e.g.,generating a plasma of ions. The electric field can have a voltage ofbetween 500 and 50,000 V, 3000 and 12000 V, or 4,000 and 8,000 V. Theelectrical field and/or ionized atoms can be generated by a fieldgenerator such as a corotron or scorotron. The second substrate can begrounded. A field generator can be scanned across the first substratewhile it is apposition to the second substrate. In one embodiment, thereis no physical contact between the first and second substrate, e.g.,there is a gap. The gap can be at least partially under vacuum.

[0086] In one embodiment, the first substrate is flexible, e.g., paperor a transparency or transparency-like surface. The second substrate canbe rigid. The positioning can include aligning the first and secondsubstrate

[0087] The method can include coupling the nucleotide to the secondsubstrate, e.g., by applying an activator compound, e.g., as describedherein. The method can further include repeating the forming,positioning, transferring, and coupling for each register of the nucleicacids to be synthesized.

[0088] In another aspect, the invention features a method of providingan array of chemical compounds. The method includes providing aplurality of interim substrates. Each interim substrate includes adifferent particles of a component of a complex chemical compound. Theparticles are disposed at different addresses of the interim substrate.The method also includes sequentially, for each interim substrate of theplurality of interim substrates, transferring the particles from thedifferent addresses of the interim substrate to corresponding addresseson a target substrate, and coupling the components to the targetsubstrate to form a nucleic acid at each of the different addresses ofthe target substrate. In the cases in which at least some of theparticles include a nucleotide, the method can be used to provide anarray of nucleic acids.

[0089] In another aspect, the invention features an apparatus thatincludes a first substrate, a second substrate, and an electrical fieldgenerator. The first substrate includes dry depositions of polymersubunits. Each deposition is at a predefined address such that differentpolymer subunits are selectively positions at different addresses on thefirst substrate. The first substrate can be, e.g., flexible, e.g., paperor a transparency. The second substrate can be flexible or rigid, andoptionally transparent, e.g., glass. The first and second substrate arepositioned in apposition to each other. In one embodiment, the secondsubstrate is grounded. The electrical field generator can be, e.g., acorotron or a scorotron. The generator can be attached to a guide ortranslator which can translate the generator across the first substrate.

[0090] In still another aspect, the invention features an article thatincludes a machine-readable medium that stores machine-executableinstruction. The instructions cause a machine (e.g., a processor) to (1)store biopolymer sequences, each of the sequences being associated witha two-dimensional address on a substrate; (2) generate layer maps,wherein each of the layer map corresponds a position in at least asubset of the biopolymer sequences and assigns monomer identities to atleast a subset of addresses of the substrate; and sequentially for eachof the layer maps, and (3) process (e.g., rasterize) the layer map togenerate triggers for each of the photoreceptors such that the triggersselectively illuminate regions of the photoreceptor, each regioncorresponding to an address of the substrate, the regions beingilluminated only if the corresponding address is assigned to the monomerfor which the photoreceptor is configured. The machine can interfacewith the charge pattern generators, e.g., the light emitters of thecharge pattern generators, to selectively trigger the light emitters toilluminate regions of the photoreceptor. The instructions can furthercause the machine to send the triggers to appropriate applicator units.

[0091] The instructions can include detecting signals from an apparatusto synchronize the position of a substrate along a substrate path with aphotoreceptor. The instructions can also include accepting input from auser, the input indicating the size of each address on the substrate.Other inputs include identifiers and/or sequences of the biopolymers tobe synthesized.

[0092] Aspects and details of the features described above can becombined in numerous ways as apparent to the skilled artisan and fromthe detailed description below.

[0093] The methods, compositions, apparati, and systems described hereprovide a method for the fabrication of arrays of biopolymers in largenumbers, in many varieties, at high speed, with great precision and lowcost. Notably, in embodiments using a software controller to directdeposition of chemical monomers, considerably versatility is achieved,as features of the array can be sized and resized based on softwarecommands without any change or adaptation of an apparatus orcomposition. Thus, for example, the same instrument can be used tofabricate dense arrays with numerous features (e.g., for high-throughputapplications) as well as sparse arrays having large features (e.g., fordetailed studies that required increased accuracy and reproducibility).

[0094] Many aspects of this disclosure are suited for the challenges ofindustrial-scale production of oligonucleotide arrays. For example, (a)they facilitate: high synthetic yield at each coupling step; (b) theyare spatially addressable on the substrate; (c) they are flexible intheir adaptation of a configuration of any specified set of sequences inany pattern; (d) they are amenable to mass production. The methods alsodo not require the usage of a photomask or a chemical mask layer, e.g.,an epoxy, lacquer, or oil mask that is stripped and reapplied betweenadditions of different compound. Chemical activation of coupling avoidsthe use of heat which is generally harsher and less energy efficient.

[0095] The details of one or more embodiments of the invention are setforth in the description below. Other features, objects, and advantagesof the invention will be apparent from the description and from theclaims.

BRIEF DESCRIPTION OF THE DRAWINGS

[0096]FIGS. 1, 2, 4, and 19 are flowcharts of exemplary processes.

[0097]FIG. 3 is a schematic of an exemplary nucleotide printing process.

[0098]FIGS. 5 and 6 are schematics of an exemplary unit for chargingnucleotide particles.

[0099]FIG. 7 is a schematic of an exemplary process for electrostaticdeposition using a photoreceptor.

[0100]FIGS. 8, 9, and 10 are schematics of exemplary apparati forprinting nucleotide particles.

[0101]FIGS. 11A and 11B are a schematic of an exemplary device forgenerating an aerosol.

[0102]FIG. 12 depicts an exemplary process for capping of unusedreactive groups.

[0103]FIG. 13 depicts the chemical structure of exemplary nucleotidemonomers.

[0104]FIG. 14 is a schematic of an exemplary transfer device.

[0105]FIGS. 15 and 16 are images of printed substrates.

[0106]FIG. 17 is a block diagram of an exemplary controller.

[0107]FIG. 18 is a schematic of an exemplary process for image mapconstruction.

DETAILED DESCRIPTION

[0108] An array of chemical compounds can be produced by theelectrostatic deposition of its components onto a substrate. The subunitare coupled to the chemical groups on the substrate to synthesize acomplex compound. By localizing the electrostatic deposition, differentbuilding blocks can be coupled at different positions on the substrate.Thus, a diverse and addressable set of chemical compounds is produced onthe substrate to form an array of chemical compounds, e.g., ofbiological polymers. One application of this concept is the productionof an oligonucleotide array. Other concepts provided here can be used incombination with the electrostatic deposition method or with otherchemical synthetic methods.

[0109] The electrostatic deposition of chemical subunits, at least insome respects, includes features of electrophotography.Electrophotography is a complex process of printing images using lightand the electrostatic force. In some implementations, it includes atleast six processes: charging a photoreceptor, expose to light to formlatent image, toner development, transfer of imaged toner, fusing of thetransferred toner and cleaning of the photoreceptor. Xerography(meaning, “dry writing” in Greek), is one common application ofelectrophotography, widely used in paper copiers and printers to producehigh quality images (see, e.g., “Xerography” by Dan A. Hays,Encyclopedia of Applied Physics, American Institute of Physics, Vol. 23,541-561, (1998) and “Physics of Electrophotography”, by Damodar M. Paiand B. E. Springett, in Review of Modern Physics, Vol. 65, No. 1,January (1993)).

[0110] As disclosed in U.S. Pat. No. 2,297,691, photoconductivematerials are charged in the dark and then exposed to patterned light inthe form of a projected image to produce an electrostatic image which isthen developed using a charged pigmented powder, often referred to as atoner. A review of the xerographic technology is provided in “ThePhysics & Technology of Xerographic Processes,” by E. M. Williams,(1984), and “Electrography and Development Physics,” by L. B. Schein,(1988).

[0111] Referring to the exemplary process shown in FIG. 1,oligonucleotide array construction includes the digital design 102 ofthe array, printing 103 the array, post-processing 104 of the printedarrays, hybridization 105 of a sample to the array, and reading 106 thearray.

[0112] The digital design 102 includes the patterning a two-dimensionalmap with unique positional addresses. The digital design process is alsodescribed below (see “Digital Design”). Each address is assigned anucleic acid sequence. Typically, different sequences are positioned atdifferent addresses, although some may be duplicated to provide controlsand verification. Referring to FIG. 18, the digital design processtranslates the set 801 of strings representing the nucleic acidsequences into a series of layer maps, 820 a, 820 b, 820 c, 820 d, eachlayer indicating a nucleotide monomer to be added at a given register. Alayer map includes information for four different image maps, 830 a, 830g, 830 c, 830 t, one for each nucleotide (A, G, C, T). Each image mapindicates, typically in a binary fashion, whether the given nucleotideis to be added to an address. Thus, a layer map for adenine indicatesaddresses where an adenine nucleotide is to be coupled to a growingchain. Each nucleotide image maps is interpreted during the printing103, e.g., to control the spatial position of electrostatic depositionof nucleotide monomer subunits, e.g., as described below.

[0113] It is also possible stagger the registers, e.g., by not adding amonomer for some addresses in a particular layer map, and it is possibleto include information for additional image maps, e.g., for unnaturalnucleotides or other chemical compounds to be added. Similarly, if thenucleic acids being synthesized are of different lengths, some addressesmay not be indicated for nucleotide deposition in at least one of thelayer maps.

[0114] Referring to FIGS. 2 and 3, a first exemplary implementation ofthe printing process 103 includes cyclically depositing the fourcanonical nucleotide compounds 111, 112, 113, 114 onto a substrate 20,and coupling 116 the depositing nucleotide molecules to a reactive endof a growing chain on the substrate 20. The deposition of the fourdifferent nucleotides typically occurs sequentially. Four applicatorunits are used, each is dedicated to the printing of one of thecanonical deoxyribonucleotides (A, G, C, T). Spatial information, e.g.,in the form of an image map (see below), indicates to each applicatorunit regions of the substrate 20 where the nucleotide compounds is to beapplied. The substrate is post-processed 117 for chemical modification.After one cycle, the process is repeated 120 until all the nucleic acidsare synthesized.

[0115] Referring to FIG. 4, a second exemplary implementation of theprinting process 103 includes: two cycles 123, 124. The cycle 123results in the printing of interim substrates whereas the cycle 124results in the synthesis of polymers on the target substrate. The cycle123 includes cyclically depositing nucleotide particles using fourapplicator units 111, 112, 113, 114 onto an interim substrate, andtransferring 115 the particles from the interim substrate to a targetsubstrate. The cycle 124 includes, after the transfer process 115,coupling 116 the transferred nucleotide molecules to a reactive end of agrowing chain on the target substrate, and post-processing 119 thetarget substrate. After one cycle, the process is repeated 120 until allthe nucleic acids are synthesized.

[0116] In the embodiment shown in FIG. 4, the cycles 123 of interimsubstrate printing are synchronized with the synthetic cycles 124 of thetarget substrate. In this case, an interim substrate is not printeduntil the deposits on a previous interim substrate have been transferredto the target substrate. In another embodiment (not shown in FIG. 4),the cycles 123 of interim substrate printing are decoupled from thesynthetic cycles 124 of the target substrate. In one example, all theinterim substrates are printed, i.e., one for each layer. Aftercompletion 121 of the interim substrate printing cycles 123, the interimsubstrates are sequentially transferred to the target substrate. Otherexamples can include varying degrees of temporal coupling between thecycles 123 and 124.

[0117] Toner Preparations

[0118] For electrostatic deposition, particles of polymer subunits arecharged and then bound to a patterned region on the surface of aphotoreceptor. When the polymer subunits are nucleotides, the particlescan be composed of nucleotides that are protected on one terminus, andactivated on the other. (The terminus refers to the nucleotide C-5′ orC-3′ position.) Exemplary nucleotide compounds I, II are diagramed inFIG. 13. Compound I has a 3′ phosphoramidite and a 5′ protecting group.Compound II has a 5′ phosphoramidite and a 3′ protecting group. In stillother examples, e.g., following the synthetic process described in U.S.Patent application Publication US2001/0044530, published Nov. 22, 2001,the compound includes a protecting group, but not the phosphoramidite.In this case, the substrate to which the nucleotide is coupled can bemodified with a phosphorous activating group such as a phosphoramidite.Other nucleotide coupling chemistries can be used, e.g., phosphodiesteror phosphotriester. Both dry and liquid toners that include thenucleotide particles can be prepared.

[0119] Nucleotide particles are produced from powders of amorphousnucleotide subunits (e.g., phosphoramidite nucleotides). The powders areprocessed (e.g., by jet milling) to produce particles. The particles aretypically less than 100, 50, 30, 20, 18, 15, 12, 10, 9, 8, 7, or 6 μm indiameter, e.g., on average about 7 μm in diameter. For example, afterjet milling, the particles can be sorted by a classifier into desiredsize ranges, e.g. less than about 30, 20, 10, or 5 microns in diameter.

[0120] The nucleotide particles are can be combined with other additivesto prepare a nucleotide toner preparation. The toner preparation is thenmixed with a carrier preparation that includes carrier beads thatinclude a metal oxide, e.g., ferrite carrier beads. One availablecarrier bead is the Powdertech carrier DM070C silicone coated ferrite.This combination, referred to as a developer composition, is thentriboelectrically charged. The carrier beads can be about, e.g., 30 to80 μm in diameter. In some cases, they are larger in diameter than thenucleotide particles. The charge characteristics, Q/M (charge over massratio) are measured with a charge spectrometer such as the “q-test”equipment made by Epping GmbH, Germany and/or Q/M meter (Model 210HS,Trek Inc., NY). It has been found that nucleotide particles of less than50 μm diameter are chargeable to an absolute value of at least between 5and 50 μC/g.

[0121] In the presence of carrier beads, the nucleoticdes particles,absent other components, can acquire a high charge which is unstable.While such particles are usable, it is also possible to prepareparticles that can be charged in a more stable manner as it isparticularly useful if the charge is stable over time, e.g., throughoutmultiple printing runs. The charging properties of the particles can becontrolled by the addition of a surface charge control agent.

[0122] A surface charge control agent is an agent that has one or moreof the following functions: stabilizing charge on the nucleotideparticles, distributing charge between nucleotide particles and otherparticles, and determining polarity of the charge on the nucleotideparticles.

[0123] By using certain surface charge control agents, the nucleotideparticles are positively charge during the mixing. Other surface chargecontrol agents can be used to negatively charge the particles. Theprinting apparatus is configured according to the polarity of the chargeon the nucleotide particles.

[0124] Referring to FIG. 5, an applicator unit is used totriboelectrically charge a toner composition and selectively bind it toa photoreceptor 41. The toner composition, which includes nucleotideparticles 52 (initially uncharged) and surface charge control agents 51,is dispense from the reservoir 50 into the chamber 48. In the chamber 48it is mixed with ferrite carrier beads 44. Rotating magnetic brushes inthe chamber 45, 46 agitate the mixture. Contact between the carrierbeads 44 and the nucleotide particles 47 results in triboelectriccharging of both. As shown in this example, the nucleotide particles arepositively charged. In the vicinity of the photorecepior 41, thepositively charged nucleotide particles 47 are attached to regions onthe photoreceptor surface that are not positively charged. These regions49 were created by selectively illuminating the photoreceptor 41 using alight source 42. See, FIG. 7 and its description for additional detail.

[0125] Similarly in FIG. 6, the toner composition becomes negativelycharged. Carrier beads that favor negative charging are combined withthe nucleotide particles and tested as described below. Beads can beobtained from Powdertech (Chiba-Ken 277-0872 Japan).

[0126] Examples of surface control agents that stabilize the nucleotideparticles (e.g., the charge and/or structural property of the nucleotideparticles) include: polystyrene, polyformaldehyde,polymethylmethacrylate, polyamide, fumed silica, amorphous silica,polyethylene glycol succinate, polyethylene glycol adipate, polydiallylphthalate, polyurethane elastomer, styrene-acrylonitrile copolymer,styrene-butadiene copolymer, polyisobutylene, borosilicate glass,polyethylene glycol terephthalate, epoxide resin, polychlorobutadiene,butadiene-acrylonitrile copolymer, polyacrylonitrile, polyethylene,chlorinated polyether, polytrifluorochloroethylene andpolytetrafluoroethylene.

[0127] By adding surface charge control agents such as cabosil, a fumedsilica, and Kynar, a highly fluorinated polymeric material, the chargestability of some nucleotide particles was maintained. Polystyreneimproved the triboelectrification with the ferrite carrier beads.Lubricants (such as zinesteric) can also be added.

[0128] Carrier coating materials can also influence the characteristicsof the developer preparation. Examples of such materials include thetriboelectric series which includes kynar (polyvinylidene fluoride),Saran F220 (vinylidene chloride-acrylonitrile copolymer), polysulfone,Cyclolac H-1000 (acrylonitrile-butadiene-styrene terpolymer),polyethylene, polystyrene, polyurethane, alkyl-substituted polyvinylpyrrolidone, diallyl phthalate and methyl methacrylate (see, e.g., U.S.Pat. No. 3,795,617).

[0129] One general method for preparing a nucleoticle toner is to screendifferent components mixed with the nucleotide particles for the abilityof the combination to function as a toner. Among the parameters that canbe varied are the nucleotide particle concentration (e.g., about 2 to99.9% pure nucleotides), the surface charge control agent and itsrelative concentration, and other additives selected from thetriboelectric series (see, e.g., Henniker, (1962) Nature, 196, 474).Different combinations, ratios, and agents can be tested, e.g.,systematically. The screening process can include measuring the Q/Mparameter and then a trial electrophotographic transfer. These tests canbe performed with or without carrier beads. Further adaptation of atoner composition can include monitoring the number of particle sizedistribution and charging properties after multiple rounds of printing.Structurally durable compositions are able to withstand many such cycleswithout deterioration in particle size or charging property.

[0130] The chemical synthetic processes described here can also useliquid toners compositions. Liquid toners can include submicron-sizedtoner particles are dispersed in a dielectric liquid. These nucleotideparticles are can be, e.g., between 50 Å and 30 μm, 100 Å and 5 μm, 50 Åand 1 μm in diameter, on average. The liquid toner is applied to thephotoreceptor and the toner particles, attracted by the electric fieldof the latent image, electrophoretically migrate to the photoreceptor(see, e.g., Clause and. Mayer: in Xerography and Related Processes, ed.Dessauer and Clark (Focal, New York 1965), Chapter 12). Thiselectrophoretic property is helpful for focusing the particles on thelatent image and, for at least this reason, is suited for high-densityarrays.

[0131] The charging behavior of the nucleotide particles in a liquidmedium may be defined by the zeta potential, which is the potentialdifference between the charge on the particle and the countercharge inthe liquid. The electrophoretic mobility of the particles isproportional to the zeta potential. In some implementations the absolutevalue of the zeta potential is about 35, 40, 50, or 60 mV.

[0132] The liquid medium can have a high volume resistivity (e.g., atleast 10⁸, 10⁹, or 10¹⁰ Ωcm, and optionally between 10⁸ and 10¹², or 10⁹and 10¹¹) so that it will not destroy the latent image on thephotoreceptor. The liquid medium can be a chemically inert,non-dissolving organic solvent, e.g., a high molecular weight aliphatichydrocarbon based solvent, such as an isoparaffin e.g., Isopar® C (andrelated products from ExxonMobil Chemical) (2,2,4 trimethylpentane).

[0133] The dry toner compositions can be a mixture, e.g., of thedifferent components described above, or, in some embodiments, it can beprepared as a composite. An example of the preparation of a dry tonercomposition is provided below.

[0134] Amino acid toners can also be made, e.g., following thesemethods.

[0135]FIG. 16 is an image of the successful electrostatic transfer ofnucleotide particles from a liquid toner composition that includednucleotide particles suspended in Isopar® (available from Exxon MobilCorp.). Liquid nucleotide toners are useful for the production of highdensity arrays.

[0136] Liquid and dry toner compositions can be distributed to users incontainers that include nucleotide toner particles. In addition todistributing the nucleotide particles alone, the compositions can alsobe distributed as mixtures that include one or more other components,e.g., one or more of a surface charge control agent, carrier bead,lubricant, and so forth. The toner particles in the packages can bechargeable, but may not necessarily be charged at a given time. Thesepackages can be used to replenish toner for a nucleic acid printingapparatus, e.g., an apparatus described herein.

[0137] Electrophotographic Printing

[0138] Referring to the example in FIG. 7, a photoreceptor 150, locatedwithin an applicator unit, is used to selectively deposit chargednucleotide particles using the electrostatic force. Of course, anyselectively chargeable surface can be used to deposit the chargednucleotide particles.

[0139] The photoreceptor surface 151 is first charged so that it has thesame charge as the nucleotide particles. In this example, the surfaceand the particles are positively charged. The photoreceptor surface 151can be charged, e.g., using a corotron or a scorotron.

[0140] After the photoreceptor is fully charged, it is selectivelyilluminated, e.g., by a scanning modulated laser or an array of lightemitting diodes (LEDs). The illumination is controlled by the digitizedpixel information provided by the image maps. Illumination decharges theselected regions on the photoreceptor. When the decharged region of thephotoreceptor passes through the loading zone where thetriboelectrically charged nucleotide developer composition resides, thepositively nucleotide particles are electrostatically attracted to theexposed portions on the photoreceptor. As a result of rotation, thephotoreceptor drum or belt advances, moving another position under theillumination source and the position bound by the nucleotide particlesinto a transfer zone where the particles are transferred to a substrate164. An electrical field, e.g., provided by a corona 166, facilitatesthe transfer to the substrate 164.

[0141] The photoreceptor can be, for example, a drum (see, e.g., FIG.10) or a belt (see, e.g., FIG. 9) stretched across drive and beltsupport rollers.

[0142] Referring to FIG. 8, typically at least four applicator units arepositioned in series along a single substrate path 70 so that each ofthe four canonical nucleotides can be printed onto the substrate. Thefour applicator units are controlled by a regulator 61 which candistribute image map information to each applicator unit and alsosynchronize the units so that that the four applicator units correctlyreference each address on the substrate to prevent overlaps and otherinfidelities in printing.

[0143] The image development/transfer processes are conductedsequentially at each of at least four applicator units 62, 64, 66 and 68using adenine phosphoramidite particles 63, thymidine phosphoramiditeparticles 65, guanosine phosphoramidite nucleotide particles 67 andcytidine phosphoramidite nucleotide particles 69 respectively. The fourapplicator units can be interfaced to each other, e.g., using thecontroller 61 which can provide synchronization signal, image mapsand/or layer maps to the individual units. The substrate (e.g., aninterim substrate or a target substrate) travels along the path 70through the four developer/transfer stations 62, 64, 66, and 68. Afterthe at least four subunits are printed, the substrate is processed 71,e.g., transferred to a target substrate in the case of an interimsubstrate, or treated for coupling of the subunits in the case of thetarget substrate itself.

[0144] A variety of different machine architectures can be used forproducing the nucleotide image transfer by layers. The full layer ofnucleotide image transfer can be accomplished in several ways. The fournucleotide images can be transferred sequentially to the substratesurface. They can also be accumulated on an intermediate roll or beltbefore transferring to the substrate surface. The images can also beaccumulated on the photoreceptor and are then transferred to thesubstrate surface. In the latter design, the photoreceptor needs to besequentially recharged, exposed and developed in the presence ofpreviously applied nucleotide particles.

[0145] Referring to FIG. 9, an exemplary implementation is the MultipleStation Single Pass (MSSP) architecture. This apparatus produced amimage by sequentially transferring adenosine 84, thymidine 85, guanosine86 and cytidine phosphoramidite 87 toner images on to a beltphotoreceptor 81. The rotating belt 81 is charged 82, exposed 83, anddeveloped 84 for each nucleotide subunit. Transfer of the nucleotideparticles on the belt 81 occurs after all four nucleotide subunitparticles are accumulated on the belt. Transfer is effected in thetransfer zone 89 where the full layer of the nucleotide image iselectrostatically transferred to the substrate, e.g., using a corotronor scorotron.

[0146] An alternative MSSP system is shown in FIG. 9 where four separateapplicator units, one each for adenosine 91, thymidine 92, guarosine 93and cytidine phosphoramidite 94, are arranged in tandem. Each unit hasits own photoreceptor 95, nucleotide particle loading zone 91, chargingdevice 97, exposure 98 and the transfer 96. When the imaging substrateis in the form of sheets, a belt transports the sheet to each stationwhere each different nucleoside powder image is transferred with acharge device. Each applicator unit can be interfaced to a commoncontroller 61 (e.g., as shown in FIG. 8 and as described below) or to acomputer system which provides image maps and/or layer maps to directprinting.

[0147] In other implementations, it is possible to generate aselectively charged surface without the use of light and/or aphotoreceptor. Such a selectively charged surface can similarly be usedto pattern nucleic acid subunits on a substrate.

[0148] Interim Substrate and Transfer

[0149] As seen above, one implementation features an interim substratethat receives particulate nucleotide compositions from applicator unitsby electrostatic deposition. The interim substrate can have the sameproperties as the target substrate (see “Target Substrates,” below).Typically, the interim substrate is composed of a pliable material thatcan be rapidly fed and processed by a conventional xerographic device.It has been found that both paper and transparencies (transparencies foroverhead projection of visual presentations such as are available fromcommercial suppliers such as 3M®. The interim substrate can be composedof, for example, Mylar™ (polyethylene terephthalate), polycarbonates, anonvolatile organic acid selected from citric acid, and/or cellulosetriacetate; polyvinylchlorides. It can include various coatings,including anti-static and/or hydrophilic coatings. For general guidance,see, e.g., U.S. Pat. Nos. 5,683,793; 5,672,424; 4,997,697; 4,956,225;and 5,897,540. For usage as an interim substrate, the “transparency” mayeither be transparent, partially-transparent or opaque.

[0150] Referring to FIG. 14, nucleotide particles on the interimsubstrate 330 are transferred to the target substrate 340 using anelectric field with a polarity that repels the charged nucleotideparticles on the interim substrate 330. The electric field can begenerated, for example, by a corotron, a scorotron, or a conductingroller.

[0151] The interim substrate 330 and the target substrate 340 arepositioned in apposition to each other. There can be a gap between thetwo. The target substrate is grounded. Then, as shown in FIG. 14, acorotron 310, which includes a ground shield 315 and a thin wire (e.g.,<100 μm in diameter) 312, is scanned 345 across the interim substrate330 while a high DC voltage (e.g., between 1000 to 10000 V-DC, e.g.,about 5000 to 8000 V-DC) is applied to the wire. The corotron generatesions of the same polarity as the wire. These ions are swept by theelectric field towards the interim substrate 330. The transferring canbe effected in a partial vacuum.

[0152] This process transfers the nucleotide particles; to the targetsubstrate 340. It is on the target substrate that the nucleotidecompounds are coupled to the termini of growing polymer chains. Ifdesired, prior to coupling, the particles can be neutralized on thetarget substrate after the transfer process, e.g., by passing through analternating electric field generated by a corotron driven by AC voltage.

[0153] To insure that nucleotide particles as patterned by thephotoreceptors on the interim substrate are added to the correctpositions on the target substrate, the interim substrate and the targetsubstrate are aligned prior to the transferring, e.g., with a toleranceof less than 5 or 1 μm. An appropriate tolerance can be based on thesize of the array features. Means for aligning the interim substrate andthe target substrate include: electrical contacts which complete acircuit when they are in apposition, a magnetic field generator andsensor, and/or mechanical male and female adaptors. In addition,alternative methods for transferring may be used, e.g., physical contactor vacuum suction (e.g., if the target substrate is porous).

[0154] Coupling

[0155] The coupling of a dry deposit of a nucleotide compound can beeffected by dissolving the deposit in a solution that includes anactivator compound. The activator compound can be. The activatorcompound can also be 5-(p-nitrophenyl)-1H-tetrazole,5-ethylthio-1H-tetrazole, another tetrazole derivative,4,5-dichloroimidazole, benzimidazolium triflate, or 4,5dicyano-imidazole. For some implementations, 5-ethylthio-1H-tetrazole isparticularly useful. The solvent, in which the activator is dissolvedcan be an organic solvent, e.g., acetonitrile, dinitriles, such assuccinonitrile, glutaronitrile, adiponitrile, pimelonitrile; diethylcarbonate; and propylene carbonate.

[0156] The activator compound can be applied as an aerosol. In somecases, it is helpful to prepare the activator compound in a liquidmedium that is a mixture of at least two solvents, e.g., at least twoaprotic solvents, an aprotic low boiling point solvent and an aprotichigh boiling point solvent, e.g., in ratios between 10:90 and 90:10 or20:80 and 80:20. Examples of high boiling point solvents that can beused include: dinitriles, such as succinonitrile, glutaronitrile,adiponitrile, pimelonitrile; diethyl carbonate; and propylene carbonate.Examples of low boiling point solvents that can be used include:acetonitrile and methylene chloride. The high boiling point solvent mayprevent evaporation of the activator compound from the aerosol whereasthe low boiling point solvent facilitates aerosolization.

[0157] The activator compound can also be applied, e.g., by immersion ina solution that includes the compound across the target substrate, bypipetting a solution that includes the compound onto the targetsubstrate, or by flowing a solution that includes the compound acrossthe target substrate.

[0158] When dry deposits of the nucleotide compounds are dissolved indroplets of activator compounds dispensed by an atomizer, the compoundsare rapidly activated and couple to the reactive groups on the targetsubstrates. This reaction results in the formation of covalent bondswith functional groups on the surface of the target substrate. Inapplying a first layer of nucleotide compounds to the target substrate,the compounds are reacted with functional groups either directly on thesurface or at the termini of linker compounds. For subsequent layers,the compounds are reacted with the terminal group of the growing nucleicacid polymer.

[0159] Conditions for the chemical coupling reactions and the dropletsize can be adjusted so that the reaction site is highly localized.

[0160] Different size sections of the target substrate or the entiretarget substrate can be contacted to the aerosol concurrently. Referringto the example in FIG. 11A, an aerosol generator 710 disperses anaerosol 712 through a single nozzle 714 onto the target substrate 720.Referring to the example in FIG. 11B, the aerosol generator 710 hasmultiple nozzles 714 that are spaced apart.

[0161] Post-processing

[0162] Following the coupling process, the target substrate is processedfor one or more of the following reactions: capping, oxidation anddeprotection.

[0163] Referring to FIG. 12, unreacted surface functional groups areblocked using capping reagents such as acetic anhydride in pyridine.When the surface functional group is a hydroxyl terminal, the cappingreaction results in an acylated group which is blocked from furtherelongation of the nucleotide sequence.

[0164] Oxidation of the trivalent phosphite group can be conducted usingan iodine solution (e.g., 0.05 M I₂ in a mixture of Py/THF/water in aratio of 5:93:2). The trivalent phosphite bond oxidizes to form a stablepentavalent phosphate bond. Subsequently, deprotection of the nucleotideprotecting group 4,4′-dimethoxytrityl (DMT) is conducted using 2%dichloroacetic acid in methylene chloride solution. Deprotectionproduces surface hydroxyl groups that are available for coupling withnext layer of deposited nucleotide compounds.

[0165] In addition to the exemplary use of DMT, the protecting groups ofthe nucleic acid subunits can include photo, acid or base labileprotecting groups. For example, the photolabile protecting group can benitroveratryloxy carbonyl (NVOC), nitrobenzyloxy carbonyl (NBOC),dimethyl dimethoxybenzyloxy carbonyl, 5-bromo-7-nitroindolinyl,o-hydroxy-α-methyl cinnamoyl; 2-oxymethylene antraquinone, andmethyl-6-nitropiperonyloxycarbonyl (MeNPOC) and(R,S)-(1-(3-methoxy-4-benzyloxy-6-nitrophenyl)-ethoxy) carbonyl(MBNPEOC). The photolabile protecting groups protect the nucleotide fromreacting with other nucleotides or other chemical species and arephotoactivatable, i.e., removable by exposure to an energy source suchas UV radiation, visible radiation, electric potentials, and electronbeams. The properties and uses of photolabile groups can be found, forexample, in McCray et al J. Amer. Chem. Soc. (1970) 92:6333; Amit et al.J. Org. Chem. (1974) 39:192; and U.S. Pat. No. 5,889,165. U.S. Patentapplication Publication US2001/0044530, published Nov. 22, 2001,describes photolabile protecting groups and methods for generating 3′protecting photolabile nucleotides.

[0166] Chemical protecting groups include: 4,4′-dimethoxytrityl (DMT),fluorenylmethoxycarbonyl (Fmoc), t-butyl esters, and t-butyl ethers,e.g., t-butoxycarbonyl (t-Boc). Chemically labile protecting groups areremovable by any suitable chemical method, such as acid hydrolysis. Forexample, Fmoc is base sensitive, whereas t-Boc and DMT are acidsensitive. Chemically labile protecting groups and chemical methods toremove these groups are described in, for example, Greene's “ProtectiveGroups in Organic Chemistry,” 2^(nd) Ed., published by John Wiley &Sons, New York, N.Y., 1991. Of course, the chemically labile group andthe chemical conditions necessary to remove it are selected such thatother linkages in the oligonucleotide, such as the connection of thefirst nacleotide to the linking group, are not affected when thechemically labile group is removed under specific chemical conditions.

[0167] Deprotection is tailored to the protecting group used.

[0168] Planar Supports for Chemical Synthesis

[0169] The target substrate, upon which nucleic acid subunits arecoupled, is, typically, a planar solid substrate that has at least aplanar surface. The surface includes reactive groups, such as carboxylgroups, amino groups, hydroxyl groups and the like. The solid surfacecan include a linker group such as polyethylene glycol or other carbonchains that have amide bonds or linked ether bonds. The linker groupscan be of sufficient length (e.g., 5 to 60 atoms) to facilitate subunitcoupling during the oligonucleotide synthesis. It usually contains 5 to60 atoms within the linker. The linker molecule includes a terminalfunctional group with which surface coupling reaction occurs.

[0170] The substrate can have a rigid structure, a semi-rigid structurewith flexible planar surface structure, or a non-rigid (e.g., pliable)structure. An example of a pliable glass substrate is Schott D 263 Tborosilicate glass (Schott Corp., Yonkers N.Y.). The substrate can becomposed of plastics, resins, silica or silica-based materials such asglass or silicon sheets. Polymeric materials such as polyvinylstyrene,polypropylene or membranous materials can also be used. Metals or metaloxides and carbons may also be used, e.g., as coatings.

[0171] In one embodiment, the substrate surface is optically transparentand has surface hydroxyl functionalities as those found on silicasurface.

[0172] A planar support can also be addressable in three dimensions. Forexample, after synthesizing compounds in a first matrix backed by asolid support, a second matrix, providing a second plane of compounds,can be layered over the first plane. The second plane of compounds canbe synthesized before or after the layering.

[0173] Array Packaging

[0174] When sheet-like substrates are used, after polymer synthesis,they can be easily diced into individual units. The units can bepackaged into a cartridge or adhered to a rigid support such as glass orother materials for scanning or other detection studies.

[0175] The substrate can have any of a variety of sizes and shapes. Forexample, from a substrate that is 216 cm×279 cm (8.5″×11″) in size, atleast 20 arrays on microscopic glass slides (1″×3″) can be synthesizedsimultaneously. In one example, the imaging technique produces at least1.5 million unique addresses of oligonucleotides on each substrate.

[0176] As a result of the system's flexibility and demand for concurrentprinting of many individual arrays, the arrays produced from a dicedsubstrate can be all identical, or can each differ, e.g., in sequencecontent or level of detail.

[0177] Chemical Synthesis

[0178] The methods and apparati described here are easily adapted forgenerally any chemical synthesis, for which, chemical constituentcompounds are available having a reactive group and a protective group.The method can be used to synthesize an array of peptides. Amino acidsubunits are applied using modified amino acid which have their aminogroup blocked with a protecting group, e.g., t-Boc. For the secondcycle, the t-Boc is removed from coupled amino acids, e.g., bydeprotection of the t-Boc group with acid, e.g., trifluoroacetic acid(TFA).

[0179] A diverse collection of chemicals can be synthesized as anaddressable array. These include compounds other than nucleic acids andpolypeptides. See, e.g., DeWitt et al. (1993) Nature 90:6909; and Buninet al. (1994) 91:4708. For example, the synthesized compounds can beformed from subunits such as 2-aminobenzophenones, natural and unnaturalamino acids, and alkylating agents.

[0180] One challenge for the synthesis of chemical compounds that arenot formed from linear changes of monomer subunits is the generation ofdiversity at different positions on a backbone compound. This challengecan be approached by depositing the backbone compound in a form modifiedwith a variety of protecting groups, each sensitive to a differentcondition (e.g., light, acid, and base). First, only one of theprotecting groups is removed. The dry deposition process is then used tofirst layer compounds for addition to a first position. Subsequently, asecond protecting group is removed, and a second layer is of compoundsis deposited in order to couple them to a different position on the samebackbone molecule. The method can be repeated, and further, each layerof compounds can themselves be modified with protecting groups.

[0181] In addition, with respect to nucleic acid synthesis, as seenabove, a variety of different coupling chemistries besidesphosphoramidite chemistry can be used. The methods can also be combinedwith subunits other than monomers, e.g., pre-formed dimers and unnaturalnucleotides.

[0182] Regarding the use of monomers as well as larger subunits, thesubunit derivatives that are used can be chosen to produce nucleic acidsof a particular orientation relative to the target substrate. Forexample, when C-3′-DMI protected, C-5′-phosphoramidite monomericnucleotides are used to perform the oligonucleotide synthesis, thecoupling is in the C-5′ to C-3′ direction where C-5′ is attached to thesolid surface and the C-3′ is at the terminal position. WhenC-5′-DMT-C-3′-phosphoramidite monomeric nucleotides are used instead,the coupling will be in the C-3′ to C-5′ direction where C-3′ isattached to the solid surface and the C-5′ remains at the terminalposition.

[0183] Software and Systems

[0184] The invention also features computer systems and software, e.g.,to design, and layout biopolymer sequences for arrays, and to control anapparatus to deposit chemical subunits on a substrate.

[0185] As shown in FIG. 19, a software can be used for one or more ofthe following procedures: selecting 902 biopolymer sequences from asequence database; associating 904 the selected sequences withparticular addresses on the target array; generating layer maps 906; anddirecting 908 array printing, e.g., by sending image maps derived fromeach layer map to applicator units.

[0186] The software can include a user interfaces that enables the userto connect to public and private databases and select individual orcollections of sequences by a variety of queries, including subjectmatter searches and expression localization information, as well assequence information. The user interface can also include sliders,buttons, and other graphical features, e.g., to allow the specificationof parameters such as feature size, reagent concentrations, and soforth. Also, the software can prompt the user for design options, e.g.,size of each address, spacing, number of addresses, sequence compositionand so forth.

[0187] Once designed, the software processes the design to generate 906layer maps 820 a, 820 b, 820 c, 820 d and to process each map (e.g., byrasters), e.g., to produce image maps that can control a light sourcethat projects a processed layer map onto a photoreceptor to produce animage for a particular monomer addition.

[0188] Referring to the example in FIG. 18, sequence information 805that includes four sequences (SEQ1, SEQ2, SEQ3, and SEQ4) isinterpreted. Each sequence is assigned 904 to an address of a targetsubstrate 810. In this example SEQ1 is assigned to the address 812 a,SEQ2 to 812 b, SEQ3 to 812 c, and SEQ4 to 812 d. Each register (REG) isused to generate 906 a layer map 820 a, 820 b, 820 c, 820 d. Forexample, with respect to the second register, the layer map 820 bindicates C A T and G at the addresses 820 a, 820 b, 820 c, 820 drespectively. This layer map 820 b is interpreted to create four imagemaps, one for each nucleotide subunit, 830 a for the adenine subunit,830 g for guanine, 830 c for cytosine, and 830 t for thymidine. Theimage maps direct 908 a corresponding applicator unit to deposit thenucleotide subunit at particular positions in order to support formationof the array. In some implementations, the image map provides a set ofrasterized triggers a scanning modulating laser or a light-emittingdiode array, in order to direct illumination to selective regions of aphotoreceptor configured for application of a particular monomer.

[0189] As the methods are adaptable for on-demand printing, they can beused to print oligonucleotide image patterns in a very flexible way.Similar to producing pages of text from a word processor to a laserprinter, the printing technique described here can be used to fabricatearrays of any desired sequence of oligonucleotides by inputs to acomputer, e.g., from a keyboard, mouse selection, computed orintelligent selection, or by a remote system, e.g., a second computernetworked to the first computer. The computer system can include varioussoftware adaptors, e.g., to download sequences from public and privatedatabases, and to identify useful oligonucleotide sequences from suchdata for inclusion on an array. Further the pattern, e.g., arrangementand size of the printed oligonucleotides can be modified according tothe intended use of the oligonucleotide array.

[0190] Features of the invention can be implemented in digitalelectronic circuitry, or in computer hardware, firmware, software, or incombinations thereof. Methods can be implemented in a computer programproduct tangibly embodied in a machine-readable storage device forexecution by a programmable processor; and method actions can beperformed by a programmable processor executing a program ofinstructions to perform functions of the invention by operating on inputdata and generating output. The invention can be implementedadvantageously in one or more computer programs that are executable on aprogrammable system including at least one programmable processorcoupled to receive data and instructions from, and to transmit data andinstructions to, a data storage system, at least one input device, andat least one output device. Each computer program can be implemented ina high-level procedural or object oriented programming language, or inassembly or machine language if desired; and in any case, the languagecan be a compiled or interpreted language. Suitable processors include,by way of example, both general and special purpose microprocessors.Generally, a processor will receive instructions and data from aread-only memory and/or a random access memory. Generally, a computerwill include one or more mass storage devices for storing data files;such devices include magnetic disks, such as internal hard disks andremovable disks; magneto-optical disks; and optical disks. Storagedevices suitable for tangibly embodying computer program instructionsand data include all forms of non-volatile memory, including, by way ofexample, semiconductor memory devices, such as EPROM, EEPROM, and flashmemory devices; magnetic disks such as, internal hard disks andremovable disks; magneto-optical disks; and CD-ROM disks. Any of theforegoing can be supplemented by, or incorporated in, ASICs(application-specific integrated circuits).

[0191] An example of one such type of computer is shown in FIG. 17,which shows a block diagram of a programmable processing system (system)410 suitable for implementing or performing the apparatus or methods ofthe invention. The system 410 includes a processor 420, a random accessmemory (RAM) 421, a program memory 422 (for example, a writableread-only memory (ROM) such as a flash ROM), a hard drive controller423, and an input/output (I/O) controller 424 coupled by a processor(CPU) bus 425. The system 410 can be preprogrammed, in ROM, for example,or it can be programmed (and reprogrammed) by loading a program fromanother source (for example, from a floppy disk, a CD-ROM, or anothercomputer).

[0192] The hard drive controller 423 is coupled to a hard disk 430suitable for storing executable computer programs, including programsembodying the present invention, and data including storage. The I/Ocontroller 424 is coupled by means of an I/O bus 426 to an I/O interface427. The I/O interface 427 receives and transmits data in analog ordigital form over communication links via a network port 441 such as aserial link, local area network, wireless link, and parallel link. Insome implementations, the I/O interface 427 also communicates directlywith at least four applicator units, e.g., via the port 442 which is insignal communication with the applicator unit for adenine addition (A);the port 443 in communication with the unit for G, port 445 incommunication with the unit for T, and port 444 in communication withthe unit for C.

[0193] One non-limiting example of an execution environment includescomputers running Windows NT 4.0 (Microsoft) or better or Solaris 2.6 orbetter (Sun Microsystems) operating systems.

[0194] Exemplary Applications

[0195] Oligonucleotide arrays can be used for a variety of applications.

[0196] Recent advances in genomics have resulted in the sequencing ofthe genomes of many prokaryotic and of several eukaryotic organisms areavailable. Two drafts of the human genome sequence have been published(International Human Genome Sequencing Consortium, “Initial Sequencingand Analysis of the Human Genome”, Nature 409, 860-921; 2001 and VenterJ. C., et al. “The Sequence of the Human Genome”; Science 291,1304-1350; 2001). Genome and transcriptome sequence information is auseful basis for analyzing information about the expression of genes,the function of gene products, and the relevance of geneticpolymorphisms. For example, gene expression profiling (see, e.g.,Lockhart D. J. and Winzler E. A; Nature 405, 827-836; 2000) can be usedto identify cells and conditions which express particular genes. Geneticpolymorphisms can be used to identify associations between the genotypeof individuals and disease susceptibility or responses to drugs (see,e.g., Kruglyak L., Nature Genetics 22, 139-144; 1999).

[0197] Nucleic acid arrays are useful for multiplex analysis of bothgene expression and genetic polymorphisms. For example, nucleic acidarrays can be fabricated by attaching or synthesizing a large number ofcDNAs or synthetic oligonucleotides on a surface at precise locations(for reviews see Gerhold D. et al., TIBS 24, 168-173; 1999 and Watson A.et al., Current Opinion in Biotechnology 9, 609-614; 1998). Upon thebinding of nucleic acid samples to specific features of the array,signals are generated and detected (Basarsky T. et al.; in “MicroarrayBiochip Technology, M. Schena, Ed., Eaton Publishing, Natick, Mass.;2000).

[0198] One type of oligonucleotide array is a primer array in which theoligonucleotides are attached by their 5′ terminus to the array solidsupport. The oligonucleotides on such arrays can be used as primers inprimer extension reactions. Assays based on primer extension can be usedfor genotyping single nucleotide polymorphisms (SNPs). (See Thomas A.Weaver, “High-throughput SNP Discovery and Typing for Genome-wideGenetic Analysis”, Trends in Genetics, December 2000).

[0199] Arrays of polypeptide compounds are useful for screening ligandsfor a target compound. The identified ligands may be a lead for drugdiscovery. Arrays of non-polymeric compounds are similarly useful fordrug discovery screens. Other arrays can be used as sensors, e.g., anenvironmental sensor or as a diagnostic tool, e.g., to detect thepresence of particular antigens.

[0200] The following specific examples are merely illustrative, and notlimitative of the scope of the invention. All publications, references,patents, and patent applications cited herein are incorporated in theirentirety by reference.

EXAMPLE 1

[0201] A solution of 0.25 M of 3′-DMT-thymidine-5′-CEP in acetonitrilewas prepared with 0.37 grams of3′-dimethoxytrityl-5′-phosphoramidite-deoxythymidine dissolved in 2 mlof distilled, anhydrous acetonitrile in a dry box. A microscopic glassslide was covalently coated with polyethylene glycol. The glass slidewas dried and stored in dry box. The3′-O-DMT-thymidine-5′-phosphoramidite solution was spotted onto theglass slide in an arrayed format at four different concentrations (25,50, 100 and 250 mM) and at different volumes (0.1, 0.25, 0.5 and 1 ul).The slide was dried in the dry box until a thin-film of3′-O-DMT-thymidine-5′-phosphoramidite developed on the glass surface.The glass slide was then dipped into a solution of 0.5 M tetrazole indry acetonitrile. The slide was washed with acetonitrile, oxidized with50 mM iodine solution (THF/Py/Water in 93/5/2 ratio) for 3 minutes. Theslide was thoroughly washed again with acetonitrile, dried and replacedin the dry box. The slide was spotted with a solution of 40 mM Cy3Amidite (Amersham Pharmacia), a fluorescence labelling reagent, inacetonitrile and 200 mM tetrazole in acetonitrile. Labelling with Cy3amidite indicates immobilized reactive groups on the substrate whichfailed to react with the thymidine deposited as a thin film.

[0202] The resulting slide was then washed with acetonitrile, methanoland SSPE buffer. The slide was inserted into a laser scanner, ScanArray(GSI Lumonics) excited at 540 nm. The percentage relative light units (%RLU)| were determined. The results demonstrated that3′-O-DMT-thymidine-5′-phosphoramidite was indeed coupled to the glassslide and the higher the compound concentration the better the couplingyield. At concentration closer to 100 mM, the coupling almost reachedsaturation. See also FIG. 14 and Example 1 of U.S. Provisional PatentApplication Serial No. 60/279,004, filed Mar. 26, 2001.

EXAMPLE 2

[0203] As described for the thin-film of3′-O-DMT-thymidine-5′-phosphoramidite compound developed on the glasssurface in Example 1, the following experiment indicated that immersionof the glass slide in 0.5 M of tetrazole solution in acetonitrile didnot cause a dissolution and diffusion of the thin-film compound into thetetrazole solution.

[0204] A 0.25 M solution containing3′-O-DMT-thymidine-5′-phosphoramidite in acetonitrile was used to spoton a glass slide which was previously coated with polyethylene glycol. Afluorescence labeling Cy3 solution was applied and the glass slide wasscanned through a laser scanner, ScanArray (made by GSI Lumonics). Ablue circle and a yellow circular background was observed. The bluecircle indicated that the spot was covered by the DMT protectedthymidine compound which was covalently attached to the blue spot. Theyellow background indicated the presence of the unreacted linkermolecules containing the free hydroxyl group which reacted with the Cy3Amidite. The clear and discrete boundary between the blue circle and theyellow background indicated that there is no diffusion of the physicallycoated 3′-O-DMT-thymidine-5′-phosphoramidite compound when the reagentspotted glass slide was immersed in the tetrazole solution. Thisexperiment demonstrated that while the spot coated thymidinephosphoramidite compound dissolved in tetrazole solution, it wasactivated instantaneously and reacted with the hydroxyl groups on theglass surface. See also FIG. 15 and Example 1 of U.S. Provisional PatentApplication Serial No. 60/279,004, filed Mar. 26, 2001.

EXAMPLE 3

[0205] The 5′-DMT-dA-(β-cyanoethyl)-phosphoramidite compound waspulverized using a jet mill and sorted using a classifier to collectpowders with different, predefined particle size. The chargecharacteristics of a processed nucleotide sample, LT-511-811-1(5′-DMT-dA-(β-cyanoethyl)-phosphoramidite), was measured using a chargespectrometer q-test, made by Epping, GmbH, Germany. This positivelycharged sample had a q/d value of 28 fC/μm.

[0206] Similar samples, L30 and L38 were used as a toner in a printengine on Kyocera model 8000 which is designed for use with positivetoners. Although usable, the triboelectric charge of these samplesdecreased after only a few prints.

[0207] To improve the charge characteristics, surface charge controlagents and one of the selected triboelectric series such as polystyrenewere used as additives. The procedure is described as follows.

[0208] 1. Hercules D125 polystyrene was jet milled to 7.8 micron averagediameter. It was then classified to obtain a preparation ofapproximately 70%>5 micron (percentage by number). The jet mill used wasthe Page: 41 [0]Trost™ TX jet mill (from Plastomer Products, Newtown)

[0209] 2. The nucleotide powder was jet milled and classified to collectthe finest particle fraction (typically less than 3 microns diameter) ina filter bag. Classifiers that can be used include: Alpine® 100 MZRClassifier (from Hosokawa Micron Power System, Summit, N.J.).

[0210] 3. Fine nucleotide particle/Silica blends were produced byblending 4 grams of fine nucleotide powders and 1 g of Degussa R504fumed silica at medium speed in a Sorvall blender.

[0211] 4. 1 gram of blended nucleotide powders/fumed silica from (step3) was blended for 2×5 sec with 40 g of polystyrene powder from step 1.

[0212] 5. 0.35% Pennwalt Kynar 461 is added to this blend and mixed 2×5sec. to produce the final nucleotide toner preparation.

[0213] 6. A “treated” carrier is prepared by mixing 3000 g Powdertechcarrier DMO70C silicone coated ferrite with 0.65 g R504 filmed silicaand 0.2 g 812 fumed silica for 1 hour (e.g., about 0.5% R504 fumedsilica; and 0.1% 812 fumed silica).

[0214] 7. A developer is prepared by blending 30 grams of the finalnucleotide toner from step 4 with 570 grams of treated carrier for 1hour.

[0215] The exemplary final toner preparation (e.g., from step 5)includes about 2% nucleotide particles; 0.5% fumed silica; 97%polystyrene powder; 0.35% Knyar. The toner preparation is then combinedwith the treated carrier to form the developer, e.g., as described insteps 6 and 7.

[0216] The resulting mixtures were then subjected to the measurement ofelectrostatic charge distribution using the q-test apparatus and theTrek Q/M device (Model 210HS). This nucleotide developer preparationmaintained a stable positive charge after triboelectric charging.

[0217] The two samples L47-A (adenyl compound) and L47-T (thymidylcompound) nucleotide developer compositions also gave stable chargecharacteristics and maintained acceptable charge even after 1200 prints.The particular qualities of these toner can be further controlled byvariation of the particular agents selected (e.g., the surface chargecontrol agent) and the ratios.

EXAMPLE 4 Printing Nucleotide Particles

[0218] An electrophotographic print machine purchased from Kyocera model8000 was modified to accommodate the positively charged nucleotidetoners, the user interface for printing specific array format. Inaddition, the physical fusion mechanism was eliminated. The fournucleotide toners, each contained one of the four nucleotide monomersdA, dT, dG and dC, were placed in toner compartments. Those fournucleotide toners were prepared according to the procedures described inExample 3. The latent image created on the photoreceptor was introducedfrom a software program designed to provide image layers converted froma set of selected oligonucleotide sequences. A set of arrays containing400,000 image spots was used as an example in the printing process. Theprinting was conducted using an interim substrate such as thetransparency sheet (e.g., Model PP 2200 and PP 2500 from 3M®). After theprinting, the nucleotide toners image was transferred to a glasssubstrate through a scanning corotron which was induced with 6,000 DCvoltage. As shown in FIG. 15, the resulting nucleotide image on theglass substrate contained the corresponding 400,000 spots and thelocation of each spot contained nucleotide powder addressed atpredefined position according to the image design layout from thesoftware program. In this case, each spot is 150 μm in diameter with apitch of 300 μm. The software program also allows one to produce spotsizes from 50 μm to 500 μm in diameter anywhere on the same substrate.The spots can be any shape, e.g., circular, elliptical, rectangular, orsquare. The flexibility to choose spot size and sequence offersadvantages for the optimization of the subsequent hybridizationexperiment.

EXAMPLE 5 Chemical Fusion

[0219] After the image transfer to the glass substrate was conducted asdescribed in Example 4, arrays of the nucleotide phosphoramidites wereready to be chemically “fused” to the substrate surface. The glasssubstrate was subjected to a moisture controlled chamber where anapparatus containing an atomizer was installed. The atomizer wasattached to a pressurized reagent bottle. Other means for making a mistinclude: a sonicator and a piezoelectric dispenser. The reagent wasprepared from a solution containing 0.4 M of tetrazole in a mixedsolvent that includes acetonitrile and propylene carbonate in a 60/40ratio.

[0220] When the pressure was applied, the atomizer produced a very finemist of the chemical reagent. The fine mist contacted the substratesurface created the reagent droplets of approx. 10 μm in diameter. Assoon as arrays of the nucleotide powders were in contact with thereagent droplets, the nucleotide powders instantaneously dissolved andactivated by the reagent droplets. The activated nucleotide thenimmediately reacted with the surface of the glass substrate. The wholeprocesses occurred within about 10 seconds after the atomizer wasapplied to the glass substrate. Coupling of the activated nucleotideswith the surface hydroxyl groups on the glass substrate formed the basisof chemical fusion of the nucleotides with the substrate. After thefusion reaction, the glass substrate was immersed in a bath of methanolto quench the unreacted reagent on the substrate surface. The substratewas washed thoroughly with methanol and dried for the next surfacechemical operations.

[0221] In order to ensure high coupling yield of the nucleotides in eachlayer, the procedure used in Example 4 and 5 can be repeated with thesame image layer on the substrate. Multiple couplings can be used toimprove the quantitative yield, although it may not be necessary

EXAMPLE 6 Capping

[0222] After the chemical fusion of the printed arrays was accomplishedas described in Example 5, the glass substrate was immersed in a reagentbath containing a mixture of 1:1 ratio of acetic anhydride and pyridinefor 5 minutes. As shown in FIG. 12, the surface hydroxyl groups outsidethe toner image area were inactivated by acylation. This created apredefined size and location of the nucleotide arrays. The arraysubstrate was then washed two times with methanol and air dried.

EXAMPLE 7 Oxidation and Deprotection

[0223] The array substrate obtained from Example 6 was immersed in abath containing oxidation reagent prepared from an iodine solution (0.05M I₂ in a mixture of Py/THF/water in a ratio of 5:93:2) for 2 minutes.The substrate was then washed thoroughly with methanol and air dried.Deprotection of the nucleotide protecting groups 4,4′-dimethoxytrityl(DMT) was conducted by immersing again the glass substrate in a bathcontaining 2% dichloroacetic acid in methylene chloride solution for 1minute. The array substrate was washed twice with methylene chloride anddried for 2 minutes in an oven maintained at 50° C. In order to examinethat the arrayed nucleotides were covalently coupled to the glasssubstrate, the glass substrate was immersed in an activated Cy3 reagentfor 20 seconds. The activated Cy3 reagent was prepared from mixing anacetonitrile solution containing 100 mg of Cy3 phos phoramidite andanother solution containing 0.4 M of tetrazole in acetonitrile. The Cy3treated glass substrate was washed with acetonitrile and subjected to alaser scanning instrument, ScanArray, made by Packard BioSciences. Thescanned image clearly indicated the presence of nucleotides covalentlyattached to the substrate surface with the same array image.

EXAMPLE 8 Synthesis of an Oligonucleotide Array

[0224] A glass slide (25×75 mm), pre-coated with polyethylene glycol (MWca. 400), was spotted with a solution of5′-dimethoxytrityl-3′-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramiditeof 2′-deoxynucleoside (0.1M in ace.tonitrile). The spotting wasperformed on a Cartesian MicroSys™ SQ 4000 System (CartesianTechnologies, Inc.) according to the manufacturer's operating manual.This device uses a solenoid to deposit solutions of phosphoramiditenucleotides. After spotting, the slide was heated at 80° C. for 5minutes to evaporate the solution and form a dry film of thephosphoramidite nucleotide. The slide was subsequently transferred to aglove box purged with nitrogen gas.

[0225] The phosphoramidite derivative of the nucleoside was coupled tothe polyethylene glycol linker on the glass slide as follows. The glassslide was sprayed with an aerosol of 0.4M tetrazole solution in anorganic solvent through a spray head for 10 seconds. The reaction wascontinued for 2 minutes at ambient temperature and the glass slide wasthoroughly washed in acetonitrile. The unreacted functional groups onthe slide were capped with a solution of 1.1M 1-methylimidazole, 0.66Macetic anhydride, and 0.54M 2,6-lutidine in tetrahydrofuran for 2minutes. The slide was then removed from glove box and washed twice withacetonitrile. The phosphite triester was oxidized with 50 mM iodine in amixture of tetrahydrofuran-pyridine-water (93:5:2) for 1 minute. Afterwashing with acetonitrile twice, the dimethoxytrityl protecting groupsof the nucleotides were removed by treating the slide with 2%dichloroacetic acid in methylene chloride for 1 minute. The slide wasthen washed with acetonitrile twice and dried at 80° C. for 5 minutes.The slide was subsequently placed on the (Cartesian system for anothercycle of spotting. These processes is repeated to synthesize thenumerous different DNA sequences on the array.

[0226] A 4×6 array with 1.5 mm spacings was constructed using 20 μlspots and the method described above. The protecting groups were removedby treating the slide with a mixture of ethylene diamine:ethanol (1:1)for 2 hours at ambient temperature. The slide was then washed withethanol, de-ionized water, acetone, and dried in air. Two differentoctamer nucleic acid sequences that differ by only a single nucleotidewere synthesized on the array. The array was then tested byhybridization with a Cy3 dye labeled probe that was exactlycomplementary to one of the two octomer sequences. The probe sequence,nucleic acid sequences on the array, and hybridization results aredetailed in U.S. application Ser. No. 60/322,362, filed Sep. 14, 2001.

[0227] The probe was hybridized to the array at 25° C. for 3 hours in ahumidity chamber. The slide was washed with 5×SSPE buffer for 1 minute,air dried, and scanned for Cy3 signals on a GSI Scan Array 4000 system(Packard Instruments). Addresses which have nucleic acids complementaryto the probe, gave bright fluorescent signals indicating a significanthybridization between the synthesized oligonucleotides and the probe.Addresses that have the mismatched sequence, on the other hand, showedlittle signal, indicating no significant hybridization. These resultsconfirm the fidelity of the synthetic process.

[0228] Similar results were obtained with a 12-mer sequence.

EXAMPLE 9

[0229] Protected nucleotide monomers, A, T, G, and C are spotted onsurface treated microscopic glass slides. Four piezo-electric ink jetnozzle heads are used to deliver the four basic nucleotide monomers todifferent addresses on the slide. The microdrops of the nucleotides aredried in the air at room temperature or in an oven heated, for example,at a temperature of less than 80° C. to form thin-films, typicallyshaped as circular spots. The size of the spotted thin-films ranges from20 nm to 1500 nm depending on the spotting volume.

[0230] The nucleotide monomers are protected with trityl groups at thedistal end and phosphoramidites on the proximal end of the nucleolidemonomers. An aerosol of 5-ethylthio-1H-tetrazole is contacted to theslide so that monomers are coupled to functional groups on the surfaceat the different addresses. The aerosol cloud can contact the entireslide at once.

OTHER EMBODIMENTS

[0231] A number of embodiments of the invention have been described.Nevertheless, it will be understood that various modifications may bemade without departing from the spirit and scope of the invention. Forexample, other embodiments of apparati described herein can includeapparati with various drums and belts inserted for transferring chemicalcompounds from the photoreceptor to the substrate. Further, as would beapparent to the artisan of ordinary skill, the methods described herecan be adapted to the synthesis of non-polymeric compounds, e.g.,addressable combinatorial chemical libraries based on a molecularscaffold. Other embodiments are within the scope of the followingclaims.

What is claimed:
 1. A composition comprising: particles of less than 50μm diameter, the particles comprising a nucleotide that includes a 5′and 3′ functional groups, wherein one of the functional groups is aprotecting group and the other is a reactive group.
 2. The compositionof claim 1 wherein the particles are at least 0.1% of the composition byweight.
 3. The composition of claim 2 wherein the particles are between1% to 95% of the composition by weight.
 4. The composition of claim 1that is devoid of nucleotide particles of greater than 50 μm indiameter.
 5. The composition of claim 1 that is devoid of nucleotideparticles of greater than 10 μm in diameter.
 6. The composition of claim1 wherein the nucleotide has a C-5′ protecting group and a C′-3′phosphoramidite.
 7. The composition of claim 1 wherein the nucleotidehas a C-3′ protecting group and a C′-5′ phosphoramidite.
 8. Thecomposition of claim 1 wherein the nucleotide is cytosine.
 9. Acomposition comprising: triboelectrically chargeable particles of lessthan 50 μm diameter, the chargeable particles comprising a nucleotide;and carrier particles.
 10. The composition of claim 9 wherein theparticles are positively or negatively chargeable to 5 to 50 μC/g. 11.The composition of claim 10 wherein the particles are positivelychargeable to 10 to 30 μC/g.
 12. The composition of claim 10 wherein theparticles are negatively chargeable.
 13. The composition of claim 10,wherein the particles are positively chargeable.
 14. The composition ofclaim 9 wherein the carrier particles comprise a metal oxide.
 15. Thecomposition of claim 14 wherein the carrier particles comprise ferrite,zinc oxide, and nickel oxide.
 16. The composition of claim 9 whereinagitation of the composition charges the nucleotide particles.
 17. Thecomposition of claim 9 wherein the particles are charged.
 18. Thecomposition of claim 17 wherein the charge has a Q/M value of between 5and 50 μC/g.
 19. The composition of claim 9 that further comprises asurface charge control agent.
 20. The composition of claim 19 whereinthe surface charge control agent is selected from the group consistingof: polystyrene, polyformaldehyde, polymethylmethacrylate, polyamide,fumed silica, cabosil, amorphous silica, a fluorinated polymer,polyethylene glycol succinate, polyethylene glycol adipate, polydiallylphthalate, polyurethane elastomer, styrene-acrylonitrile copolymer,styrene-butadiene copolymer, polyisobutylene, borosilicate glass,polyethylene glycol terephthalate, epoxide resin, polychlorobutadiene,bultadiene-acrylonitrile copolymer, polyacrylonitrile, polyethylene,chlorinated polyether, polytrifluorochloroethylene andpolytetrafluoroethylene.
 21. The composition of claim 20 wherein thesurface charge control agent is cabosil or fumed silica.
 22. Thecomposition of claim 19 wherein the surface charge control agentcomprises a fluorinated polymer.
 23. The composition of claim 9 whereinthe composition is a dry composition.
 24. A composition comprising:particles of less than 30 μm diameter, the particles comprising anucleotide; means for controlling surface charge on the particles; andmeans for triboelectrically charging the particles.
 25. A compositioncomprising: chargeable particles of less than 15 microns diameter, theparticles include a nucleotide compound that has a phosphoramidite groupand a protecting group; and chargeable carrier beads of at least 30microns diameter.
 26. A composition that comprises: a dielectric liquidcomponent and particles, wherein the particles are insoluble in theliquid and are composed of a protected nucleotide subunit.
 27. Thecomposition of claim 26 wherein the nucleotide particles of thecomposition are less than 10 μm.
 28. The composition of claim 26 whereinthe dielectric liquid has resistivity of between 10⁸ Ωcm and 10¹² Ωcm.29. The composition of claim 26 wherein the dielectric liquid is achemically inert organic solvent.
 30. The composition of claim 29wherein the solvent is a high molecular weight aliphatic hydrocarbonsolvent.
 31. The composition of claim 30 wherein the solvent isisoparaffin.
 32. The composition of claim 26 that further comprises ameans for stabilizing charge.
 33. The composition of claim 26 whereinthe particles are between 2 and 26% of the composition, weight tovolume.
 34. A method comprising: providing a solid composition composedof a nucleotide monomer subunit that is protected at one terminus;processing the solid composition to produce nucleotide particles thatare less than 50 μm in average diameter; and combining the nucleotideparticles with a carrier agent to produce a developer composition. 35.The method of claim 34 wherein the nucleotide monomer subunit isactivated at the other terminus.
 36. The method of claim 34 wherein theprocessing comprises jet milling.
 37. The method of claim 34 wherein theprocessing comprises isolating particles that are less than 10 μm indiameter.
 38. The method of claim 34 wherein the carrier agent comprisesferrite carrier beads.
 39. The method of claim 34 further comprising,combining the nucleotide particles with a surface charge control agent.40. The method of claim 39 wherein the surface charge control agentincludes one or more of: cabosil, fumed silica, a fluorinated polymer,and polystyrene.
 41. The method of claim 34 wherein the processingproduces nucleotide particles that are less than 10 μm in averagediameter.
 42. A method comprising: providing a composition thatcomprises: (a) chargeable particles of less than 50 microns diameter,the particles include a nucleotide compound that has a phosphoramiditegroup and a protecting group, and (b) chargeable carrier beads;triboelectrically charging the nucleotide particles; contacting thecharged nucleotide particles to a selectively charged surface; andtransferring the nucleotide particles from the surface to a substrate.43. The method of claim 42 wherein the selectively charged surfacecomprises discrete areas that are electrostatically repel the chargednucleotide particles and other areas to which the charged nucleotideparticles attach.
 44. The method of claim 42 wherein the selectivelycharged surface is cylindrical.
 45. The method of claim 42 wherein theselectively charged surface is the surface of a belt.
 46. The method ofclaim 42 wherein the selectively charged surface is a photoreceptor. 47.The method of claim 41 wherein the charging charges the nucleotideparticles to between 5 μC/g to 50 μC/g.
 48. The method of claim 41wherein the composition further comprises (c) a surface charge controlagent.